Load cells with DCFDA (try 5, 10, 50 and ÁM) for 30 min
Dilute the DCFDA in HBS
Add H2O2 or pyocyanin to one sample

Then it depends on how you like to analyse. You can lyse your cells and measure the flourescence in a microplate reader. If doing so, chack protein concentration in advance and use equal amounts. Or you can analyse your cells by FACS. If you have any questions let me know.

I try again with pre-loading DCFH in different concentrations for 30 min. I use fluorescence microscope to observe the glass slides.
but it is still very weak like before.
If you have done this experiment, could you please give me your detail protocol?

Hi,
Ive been trying to do the ROS detection assay using H2DCFDA and cm=H2DCFDA. But so far not been successful. Iwant to use flowcytometry for ROS detection after treating cells with H2O2.
But the untreated and treated cells both have similar MFI in flow cytometry.
Please let me know a good protocol if you have been successful in ROS detection asay . I use WEhi231 a suspension cell line.
Thank you.